PROJECT SUMMARY DNA replication is accomplished by highly regulated macromolecular complexes called replisomes. Numerous challenges such as damaged DNA templates, repetitive sequences, and transcriptional complexes hinder DNA replication and stall replication forks. Stalled forks activate the replication stress response to signal repair of damaged DNA, recruit proteins, and halt cell cycle; thereby stabilizing replication forks and preventing collapse into toxic double strand breaks. Failure to deal with replication stress can cause diseases characterized by abnormal development, accelerated aging and cancer predisposition. For these reasons, it is critical to understand genome maintenance pathways that promote accurate DNA replication in the context of replication stress. To gain further mechanistic insight into replication stress response pathways, I previously developed a quantitative proteomic screening approach to study the protein composition of replisomes at unperturbed and stalled replication forks in human cells. This work provides evidence for checkpoint-independent replisome stabilization, a model contrary to prevailing models of replisome dissociation when checkpoint is inhibited, as well as mechanistic insights into ATR inhibitors that are currently utilized in clinical trials as chemotherapeutics. I also generated comprehensive proteomic datasets of active, stalled, and collapsed replication forks. These datasets include potential regulators of replication stress response, and also identifies mediators of replication fork protection, which is relevant to acquired chemoresistance in breast cancers. My lab will utilize this vast resource in combination with detailed functional studies to reveal previously unknown genome maintenance mechanisms. The proposed research program is designed to address three key areas in my lab: 1) Characterization of new replication stress response proteins, 2) Replication fork protection in breast cancers, and 3) Regulation of replication stress response by phosphorylation. Completion of these goals will reveal new fundamental insights into the molecular mechanisms of replication stress response that can lead to paradigm-shifting discoveries thereby expanding our view on genome maintenance mechanisms that govern DNA replication and providing avenues for designing effective treatments in cancer chemotherapy.